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PubMed1990

Differential effects of the cytoplasmic domains of cell adhesion molecules on cell aggregation and sorting-out.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that play important roles in morphogenesis and histogenesis, particularly in defining discrete borders between cell populations. Previous studies have suggested that the cytoplasmic domains of CAMs play a significant role in their adhesion properties. These domains may also be involved in regulating other cellular interactions, such as those involved in the sorting-out of cells to form tissues. In the present studies, we have compared the effects of replacing the cytoplasmic domain of one CAM with that of another CAM of different homophilic binding specificity on cell adhesion and cell sorting-out. The molecules studied were liver CAM (L-CAM) and the neural CAM (N-CAM) sd polypeptide. One cDNA was constructed that encodes a chimeric molecule composed of the extracellular domain of L-CAM and the cytoplasmic plus transmembrane domains of the sd polypeptide of chicken N-CAM (called L/N-CAM). Another was constructed encoding a truncated L-CAM missing the last 50 residues of the cytoplasmic domain. Permanently transfected lines of mouse L cells were obtained expressing the truncated L-CAM ("L-L-50 cells") or the chimeric L/N-CAM ("L-L/N cells") and were compared with cells expressing intact L-CAM ("L-L cells"). Immunoblotting and ELISA analyses demonstrated that these various cell lines expressed similar amounts of CAMs at the cell surface. Aggregation of L-L and L-L/N cells occurred at similar rates in short-term aggregation assays and was inhibited by antibodies to the extracellular L-CAM binding domain. In contrast, L-L-50 cells did not aggregate. Incubation of transfected cells with cytochalasin D, which disrupts microfilaments, markedly inhibited aggregation of L-L cells but had no effect on L-L/N cell aggregation. Mixed L-L and L-L/N cells co-aggregated in short-term assays; in the longer-term sorting-out assays, however, they behaved differently: L-L cells sorted out from both L-L/N and untransfected cells, whereas L-L/N cells did not sort out from untransfected cells. These studies not only suggest that interactions of cytoplasmic domains of different CAMs with the cytoskeleton can modulate cell adhesion but also suggest that specific interactions with certain cytoskeletal components are required for events such as cell sorting and cell patterning.

Animals↗
PubMed1997

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail.

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.

Amino Acid Sequence↗
PubMed1998

Restorer genes for different forms of Brassica cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several mitochondrial genes.

The oilseed rape plant, Brassica napus, possesses two endogenous male sterile cytoplasms, nap and pol. Previous studies have shown that nuclear restoration of pol cytoplasmic male sterility (CMS) is conditioned by a gene, Rfp, that is also involved in modifying transcripts of the pol CMS-associated orf224/atp6 mtDNA region. We now find that the nap nuclear restorer gene Rfn apparently is identical to Mmt, a gene that conditions the modification of transcripts from several different mtDNA regions, including one that is associated with nap CMS and contains orf222, a chimeric gene related to orf224. Mmt, in turn, is found to be allelic to Rfp, suggesting that restorer genes for the two cytoplasms represent different alleles or haplotypes of a single nuclear locus. This view is supported by restriction fragment length polymorphism mapping studies that indicate that Rfn and Rfp map to the same chromosomal position. Thus, in contrast to CMS in other species, different forms of Brassica CMS are restored by alleles of a single nuclear locus, and the restoration properties of these alleles reflect their involvement in the modification of transcripts of corresponding CMS-associated mtDNA regions. A survey of 51 varieties from 8 Brassica and Sinapis species failed to find evidence of Rfn(Mmt) in other than fertility-restored, nap cytoplasm B. napus. This suggests that Rfn(Mmt) arose in Brassica with nap cytoplasm and that the necessity for fertility restoration may have provided the selective pressure for its origin and maintenance.

Journal Article↗
PubMed1998

Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex.

We sublocalized the yeast nucleoporin Nup82 to the cytoplasmic side of the nuclear pore complex (NPC) by immunoelectron microscopy. Moreover, by in vitro binding assays we showed that Nup82 interacts with the C-terminal region of Nup159, a yeast nucleoporin that previously was also localized to the cytoplasmic side of the NPC. Hence, the two nucleoporins, Nup82 and Nup159, form a cytoplasmically oriented subcomplex that is likely to be part of the fibers emanating from the cytoplasmic ring of the NPC. Overexpression of Rss1/Gle1, a putative nucleoporin and/or mRNA transport factor, was shown previously to partially rescue depletion of Nup159. We show here that overexpression of Rss1/Gle1 also partially rescued depletion of Nup82. Depletion of either Nup82, Nup159, or Rss1/Gle1 was shown previously to inhibit mRNA export. As was reported previously for depletion of Nup159 or of Rss1/Gle1, we show here that depletion of Nup82 has no detectable effect on classical nuclear localization sequence-mediated nuclear import. In summary, the nucleoporins Nup159 and Nup82 form a cytoplasmically oriented subcomplex of the NPC that is likely associated with Rss1/Gle1; this complex is essential for RNA export, but not for classical nuclear localization sequence-mediated nuclear protein import.

Fungal Proteins↗
PubMed1995

A cytoplasmic tyrosine is essential for the basolateral localization of mutants of the human nerve growth factor receptor in Madin-Darby canine kidney cells.

Deletion of 58 internal amino acids from the C-terminal cytoplasmic domain of p75 human nerve growth factor receptor (hNGFR) changes its localization from apical to basolateral in transfected Madin-Darby Canine Kidney (MDCK) cells (Le Bivic, A., Sambuy, Y., Patzak, A., Patil, N., Chao, M., and Rodriguez-Boulan, E. (1991) J. Cell Biol. 115, 607-618). The mutant protein, PS-NGFR, also shows a dramatic increase in its ability to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to localize and characterize its basolateral and endocytic sorting signals. Both signals reside in the proximal part of the PS cytoplasmic tail, between positions 306 and 314. Transferring the cytoplasmic tail (19 residues) and transmembrane domain of a truncated PS mutant to the ectodomain of the placental alkaline phosphatase, an apical glypiated ectoenzyme, redirected it to the basolateral membrane and the endocytic compartments. A tyrosine at position 308, present in this short cytoplasmic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK cells. Their ability to endocytose NGF was reduced with the alanine mutant showing the stronger diminution. The PS mutant contains a short cytoplasmic sequence necessary both for basolateral targeting and endocytosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.

Alkaline Phosphatase↗
PubMed1995

The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity.

Integrin affinities for ligands can change markedly via a process termed inside-out signaling. We expressed several truncations of the beta 3 cytoplasmic domain in conjunction with an "activating" alpha subunit chimera, alpha IIb alpha 6B. Deletion of the 4 C-terminal residues of the beta 2 tail blocked inside-out signaling as assessed by the binding of an activation-specific antibody, PAC1. Several additional truncations remained in the low affinity state, but complete truncation (beta 3 delta 717) caused PAC1 binding. Activation by this truncation mutant did not depend on the alpha subunit cytoplasmic domain and was resistant to inhibitors of cellular metabolism and the over-expression of an isolated beta 3 cytoplasmic domain. Since deletion of beta 3(Leu717-Asp723) results in a constitutively activated integrin, this membrane-proximal seven amino acids of the beta 3 cytoplasmic domain is required to maintain alpha IIb beta 3 in a default low affinity state. The amino acid sequence of this region is conserved among integrins. Moreover, the conserved membrane-proximal sequence in alpha subunit tails seems to serve a similar function. Consequently, the conserved membrane-proximal regions of both integrin cytoplasmic domains control the ligand binding affinity of the extracellular domain.

Amino Acid Sequence↗
PubMed1995

The cytoplasmic and N-terminal transmembrane domains of cytochrome P450 contain independent signals for retention in the endoplasmic reticulum.

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Alkaline Phosphatase↗
PubMed1995

Cytotoxic T lymphocyte-associated molecule-4, a high-avidity receptor for CD80 and CD86, contains an intracellular localization motif in its cytoplasmic tail.

CD28 and CTLA-4, T cell receptors for B7-1 (CD80) and B7-2 (CD86) molecules on antigen-presenting cells, transmit costimulatory signals important for optimal T cell activation. Despite sharing sequence homology and common ligands, these receptors have distinct binding properties and patterns of expression. The function of CTLA-4 during T cell activation is not well understood, although an important role is suggested by complete amino acid sequence conservation of its cytoplasmic tail in all species studied to date. We report here a role of the cytoplasmic tail of CTLA-4 in regulating its subcellular localization and cell surface expression. In activated human peripheral blood T cells, or in several transfected or transduced cell types, CTLA-4 is not primarily a cell surface protein, but rather is localized intracellularly in a region which overlaps the Golgi apparatus. Transfer of 11 cytoplasmic residues, 161TTGVYVKMPPT, from the CTLA-4 cytoplasmic tail to the homologous position in CD28 was sufficient to confer intracellular localization. Mutation of the tyrosine residue (Tyr165) in this motif to phenylalanine resulted in increased surface expression of CTLA-4. Thus, the subcellular localization of CTLA-4 is controlled by a tyrosine-containing motif within its cytoplasmic domain. Contained within this motif is a binding site for SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase.

Abatacept↗
PubMed1997

Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain.

The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca2+-binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.

Amino Acid Sequence↗
PubMed1998

Internalization of the lymphocytic surface protein CD22 is controlled by a novel membrane proximal cytoplasmic motif.

CD22 is a key receptor on B-lymphocytes that modulates signaling during antigenic stimulation. We have defined a novel cytoplasmic motif in human CD22 that controls its unusually rapid turnover at the plasma membrane. Chimeric and mutated CD22alpha cDNA vectors were constructed and stably transfected in CD22-negative Jurkat T-lymphocytic cells. Two assays were employed to measure CD22alpha internalization: first, cytoplasmic uptake of radioiodinated anti-CD22 monoclonal antibody; and second, lethal targeting of a toxin, saporin, into cells via CD22 using bispecific F(ab')2 ([anti-CD22 x anti-saporin]) antibody. Results showed that CD22alpha lacking a cytoplasmic tail was not internalized and that replacement of the cytoplasmic tail of CD19 with that of CD22alpha resulted in a chimeric molecule that behaved like CD22alpha and internalized rapidly. Step-wise deletion of the cytoplasmic tail of CD22alpha located the internalization motif to a polar region of 11 residues (QRRWKRTQSQQ) proximal to the plasma membrane, a part of the molecule predicted to form a coil or turn structure. Interestingly, additional CD22 mutants showed that the two glutamine residues sandwiching the serine are critical to internalization but that the serine itself is not.

Antigens, CD↗
PubMed1998

Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail.

Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.

Amino Acid Sequence↗
PubMed2000

Evidence for long range allosteric interactions between the extracellular and cytoplasmic parts of bacteriorhodopsin from the mutant R82A and its second site revertant R82A/G231C.

Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/G231C. (i) In the double mutants R82A/G72C and R82A/A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay (tau approximately 8 ms), in R82A it occurs already during the first phase of the M-rise (tau < 1 microseconds). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical G-protein-coupled receptors.

Allosteric Regulation↗
PubMed2006

Cytoplasmic IRE1alpha-mediated XBP1 mRNA splicing in the absence of nuclear processing and endoplasmic reticulum stress.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1alpha to activate its protein kinase and endoribonuclease activities. Activated IRE1alpha cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5' and 3' fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1alpha and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F(v)-IRE1alphaDeltaN was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1alpha dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.

Animals↗
PubMed1987

Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix.

The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells. By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution. The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers. The particulate association was not affected by the presence of nonionic detergent. To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure. A portion of the CDC28 product was found to be tightly associated with these detergent-insoluble cytoplasmic matrices by both immunofluorescence and immunoblotting procedures. Although, for technical reasons, precise quantitation was not possible, it is estimated that a minimum of 2-15% of the total CDC28 product pool is involved in the association with the insoluble matrix. Alcohol dehydrogenase, a soluble cytoplasmic protein, was found not to be associated with the cytoplasmic matrices at any detectable level, whereas, in contrast, approximately 10-40% of the total cellular actin, a bonafide cytoskeletal protein, was present in these structures. The proportion of CDC28 gene product associated with the particulate fraction, and perhaps the insoluble matrix, appears to be substantially decreased during the preparation of spheroplasts.

Cell Compartmentation↗
PubMed1993

Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI).

Evidence is accumulating that the rho family, a member of the ras p21-related small GTP-binding protein superfamily, regulates cell morphology, cell motility, and smooth muscle contraction through the actomyosin system. The actomyosin system is also known to be essential for cytoplasmic division of cells (cytokinesis). In this study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein, named rho GDI, its stimulatory GDP/GTP exchange protein, named smg GDS, and botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in the cytoplasmic division using Xenopus embryos. The sperm-induced cytoplasmic division of Xenopus embryos was not affected by microinjection into the embryos of either smg GDS or the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of rhoA p21, one member of the rho family, but completely inhibited by microinjection of rho GDI or C3. Under these conditions, nuclear division occurred normally but the furrow formation, which was induced by the contractile ring consisting of actomyosin just beneath the plasma membrane, was impaired. Comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 prevented the rho GDI action. Moreover, the sperm-induced cytoplasmic division of Xenopus embryos was inhibited by microinjection into the embryos of the rhoA p21 pre-ADP-ribosylated by C3 which might serve as a dominant negative inhibitor of endogenous rho p21. These results indicate that rho p21 together with its regulatory proteins regulates the cytoplasmic division through the actomyosin system.

ADP Ribose Transferases↗
PubMed1995

A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes.

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.

Amino Acid Sequence↗
PubMed1997

ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.

The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a novel protein, ICAP-1 (integrin cytoplasmic domain- associated protein-1), which binds to the 1 integrin cytoplasmic domain. The interaction between ICAP-1 and beta1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic domains of other beta integrins, and requires a conserved and functionally important NPXY sequence motif found in the COOH-terminal region of the beta1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY motif and a Val residue located NH2-terminal to this motif are critical for the ICAP-1 binding. Two isoforms of ICAP-1, a 200-amino acid protein (ICAP-1alpha) and a shorter 150-amino acid protein (ICAP-1beta), derived from alternatively spliced mRNA, are expressed in most cells. ICAP-1alpha is a phosphoprotein and the extent of its phosphorylation is regulated by the cell-matrix interaction. First, an enhancement of ICAP-1alpha phosphorylation is observed when cells were plated on fibronectin-coated but not on nonspecific poly-L-lysine-coated surface. Second, the expression of a constitutively activated RhoA protein that disrupts the cell-matrix interaction results in dephosphorylation of ICAP-1alpha. The regulation of ICAP-1alpha phosphorylation by the cell-matrix interaction suggests an important role of ICAP-1 during integrin-dependent cell adhesion.

3T3 Cells↗
PubMed1998

Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein.

Cytoplasmic dynein, a minus end-directed, microtubule-based motor protein, is thought to drive the movement of membranous organelles and chromosomes. It is a massive complex that consists of multiple polypeptides. Among these polypeptides, the cytoplasmic dynein heavy chain (cDHC) constitutes the major part of this complex. To elucidate the function of cytoplasmic dynein, we have produced mice lacking cDHC by gene targeting. cDHC-/- embryos were indistinguishable from cDHC+/-or cDHC+/+ littermates at the blastocyst stage. However, no cDHC-/- embryos were found at 8.5 d postcoitum. When cDHC-/- blastocysts were cultured in vitro, they showed interesting phenotypes. First, the Golgi complex became highly vesiculated and distributed throughout the cytoplasm. Second, endosomes and lysosomes were not concentrated near the nucleus but were distributed evenly throughout the cytoplasm. Interestingly, the Golgi "fragments" and lysosomes were still found to be attached to microtubules. These results show that cDHC is essential for the formation and positioning of the Golgi complex. Moreover, cDHC is required for cell proliferation and proper distribution of endosomes and lysosomes. However, molecules other than cDHC might mediate attachment of the Golgi complex and endosomes/lysosomes to microtubules.

Animals↗